PROJECT SUMMARY Mycobacteria are the exception to many rules for bacteria. Their cellular structure and the way they grow defy common assumptions. Moreover, their exceptional cell biology is believed to underlie exceptional behaviors such as tolerance to drugs. Drug tolerance is general to mycobacteria, but is especially relevant to the eradication of pathogenic species such as Mycobacterium tuberculosis, which causes tuberculosis. The unique characteristics of mycobacteria could offer attractive avenues for more effectively targeting them in infection. For example, their cell envelope is a multilayer cellular structure that is one of the most complex in the bacterial kingdom and contributes to the inherent resistance of mycobacteria to antibiotics. However, very little is known about how the cell envelope is made during growth and division. In particular very little is known about the assembly of the outer membrane-like barrier known as the mycomembrane, which is a defining feature of mycobacteria due to its unusual composition and extreme hydrophobicity. The rationale of this proposal is that uncovering such fundamentals is essential to understanding mycobacterial survival in the challenging environments that they encounter. The objective of this research program is to uncover mechanisms of mycomembrane biogenesis and the connections to cell elongation. The proposal addresses three questions: Q1: How do mycobacteria export lipids to the mycomembrane? Q2: How do mycobacteria synthesize the multiple layers of the cell envelope and achieve localized growth from their poles? Q3: How do we identify the machinery for essential processes within the mycobacterial cell envelope? The PI and her team will address these questions through a combination of microbiology, biochemistry, and chemical biology. A network of collaborations supports this program. The impact of this project is to provide (1) opportunities for the rational targeting of cell envelope biogenesis for anti-infective therapy and (2) much- needed tools to investigate mycobacterial cell envelope proteins.